Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure called the root nodule, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant is most often of the family Leguminosa. Previously, Rhizobium species were informally classified into two groups, "fast-growing" or "slow-growing," to reflect their relative growth rates in culture. The group of "slow-growing" rhizobia has recently been reclassified as a new genus, Bradyrhizobium (D.C. Jordan (1982) Int. J. Syst. Bacteriol. 32:136; Bergey's Manual of Determinative Bacteriology, Vol. I, 1984, Holt et al., eds). The fast-growing rhizobia include Rhizobium trifolii, R. meliloti, R. leguminosarum, and R. phaseolus. These strains generally display narrow host ranges. Fast-growing R. japonicum (now known as R. fredii) which nodulates wild soybean and Glycine max cv. Peking but forms ineffective nodules on commercial soybean cultivars, has also been described. These R. japonicum (R. fredii) strains, as well as fast-growing members of the cowpea Rhizobium (now R. loti), display a broader host range. The genus Bradyrhizobium includes the commercially important soybean-nodulating strains of Bradyrhizobium japonicum (i.e., USDA 110 and USDA 123) and the symbiotically promiscuous rhizobia of the "cowpea group." Also included is Bradyrhizobium sp. (Parasponia) (formerly Parasponia Rhizobium) which nodulates a number of tropical legumes including cowpea and siratro, and is distinguished by its ability to nodulate the non-legume Parasponia.
Nodulation, the development of effective symbiosis, and nitrogen fixation are complex processes requiring both plant and bacterial genes. Several reviews of the genetics of the Rhizobium-legume interaction are found in W. J. Broughton (ed.) (1982) Nitrogen Fixation, Volumes 2 and 3, Clarendon Press, Oxford; A. Puhler (ed.) (1983) Molecular Genetics of the Bacteria-Plant Interaction, Springer-Verlag, Berlin; A. Szalay and R. Legocki, (eds.) (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction, Cornell University Press, Ithaca, New York; S. Long (1984) in Plant-Microbe Interaction Volume I, T. Kosuge and E. Nester (eds.), McMillan, New York, pp. 265-306; and D. Verma and S. Long (1983) International Review of Cytology (Suppl. 14) K. Jeon (ed.), Academic Press, p. 211-245.
In the fast-growing species, the genes required for nodulation and nitrogen fixation are located on large Sym (symbiosis) plasmids. Such genes include those required for nodule initiation and development (nod), those genes which have a Klebsiella pneumoniae homologs (nif), such as the structural genes for nitrogenase (nifHDK) and the regulatory gene nifA, and other genes involved in nitrogen fixation (fix). A number of genes determining nodulation functions (nodEFDABC) and the nifHDK genes have been mapped on the 180 kb Sym plasmid of R. trifolii ANU843 (P. Schofield et al. (1983) Mol. Gen. Genet. 192:459; P. Schofield et al. (1984) Plant Mol. Biol. 3:3). Nodulation and nitrogenase genes have also been mapped to symbiotic plasmids in R. leguminosarum (Downie et al. (1983) Mol. Gen. Genet. 190:359) and in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445).
The nitrogenase and nodulation genes of B. japonicum and Bradyrhizobium sp. (Parasponia) are believed to be chromosomally encoded. Sym plasmids have not been found to be associated with nitrogen fixation in the slow-growing rhizobia. A review of the genetics of symbiotic nitrogen fixation in B. japonicum is given in H. Hennecke et al. (1987) in Molecular Genetics of the Plant-Microbe Interaction, D. Verma and N. Brisson (eds.), Martinus Nijhoff Publishers, The Netherlands, pp. 191-196.
Ferredoxins are cysteine-residue-containing non-heme iron-sulfur proteins which serve as electron carriers in a variety of metabolic reactions (R. Thauer et al. (1982) in Iron Sulfur Proteins, T. Spiro (ed.), pp. 329-341). Ferredoxins are found in a number of microorganisms as well as in plants such as spinach and parsley. A ferredoxin has been isolated from Rhizobium japonicum (now Bradyrhizobium japonicum) bacteroids of soybean root nodules (K. Carter et al. (1980) J. Biol. Chem. 255:4213). The authors report that the bacteroid ferredoxin is capable of functioning as an electron donor for nitrogenase in R. japonicum bacteroides. The amino acid composition but not the amino acid sequence of the ferredoxin protein was disclosed. Thus, Carter et al. presents no teaching suggestions which would lead the skilled artisan to the present invention. Genes encoding rhizobial ferredoxins have only recently been described in the literature.
There have been reports concerning the ferredoxin genes of some nitrogen-fixing bacteria other than Rhizobium. M. Graves et al. (1985) Proc. Nat. Acad. Sci USA 82:1653, reported the sequence of a Clostridium pasteurianum ferredoxin gene, but did not suggest a nitrogenase function for it. The in vitro transcription of the C. pasteurianum ferredoxin gene has also been described (M. Graves et al. (1986) J. Biol. Chem 261:11409). The first report of linkage between a structural gene for nitrogenase and a ferredoxin gene was in Azotobacter chrococcum (R. Robson et al. (1986) EMBO J. 5:1159). Those authors also provided the nucleotide sequence of the ferredoxin gene.
Nucleotide sequences for genes identified as rhizobial ferredoxins have only recently been disclosed in the literature. P. Gronger et al. (1987) Nucleic Acids Res. 15:31 (co-authored by the inventors of the parent application hereof, Ser. No. 019,043) presented an amino acid sequence corresponding to the fixX gene of R. meliloti, and DNA and amino acid sequences for the corresponding region of R. leguminosarum. The R. leguminosarum sequence did not contain the complete ferredoxin diagnostic pattern, and the article contained no teaching suggesting that those sequences encoded or comprised ferredoxins or were useful in nitrogenase systems. C. Earl et al. (1987) J. Bacteriol. 169:1127 disclosed the ferredoxin-like sequence of the gene called fixX from R. meliloti 1021. As reported therein, the existence of this sequence was disclosed to the authors by one of the co-inventors of the parent application hereof. Similarly, I. Dusha et al. (1987) J. Bacteriol. 169:1403-1409, in an article published after the filing date of said parent application, reported the existence of the fixX gene of R. meliloti 41 and that insertional inactivation of the fixX gene resulted in a Fix.sup.- phenotype. The DNA and the deduced amino acid sequences of the fixX gene led the authors to the conclusion that fixX encoded a ferredoxin. The DNA sequence of a R. trifolii gene (termed fixX herein) and the deduced amino acid sequence of its ferredoxin-like gene product were disclosed in S. Iismaa and J. M. Watson (1987) Nucleic Acids Res. 15:3180, published after the filing data of the parent application hereof. The fixX genes of the present invention correspond to those designated "ORF1" in the parent application hereof. W. J. Buikema et al. (1987) J. Bacteriol. 169:1120-1126, in an article published after the filing date of the parent application hereof, noted that the deduced amino acid sequence of an open reading frame downstream of the R. meliloti nifB gene exhibited significant homology to the amino acid sequences of other bacterial ferredoxins; this open reading frame corresponds to the fixY gene of this application, and to ORF2 of the parent application hereof. J. Noti et al. (1986) J. Bacteriol. 167:774-783, disclosed the DNA sequence of nifB gene and about 81 bp downstream from the nifB stop codon. Within that sequence downstream of nifB is found the DNA encoding approximately the first 72 bp of the frxA gene of the present invention, including the sequence coding for the diagnostic ferredoxin pattern of cysteine residues. However, Noti et al. (supra) give no teaching suggesting that there is a ferredoxin-like gene in that segment of Bradyrhizobium DNA. Furthermore, this article teaches away from the present invention because Noti et al. proposed that downstream of nifB there is an 831 bp ORF, starting with an ATG translation initiation codon about 8 bp 3' to the nifB stop codon. The work of the present invention teaches that the frxA ferredoxin gene is a 222 bp ORF which begins with a GTG translation initiation codon about 10 downstream of the nifB stop codon. Furthermore, the reading frame of frxA is different from that of the ORF disclosed in Noti et al. (supra). Information concerning frxA was disclosed in S. Ebeling et al. (1988) J. Bacteriol. 170:1999-2001, an article co-authored by one of the co-inventors of this application.
Ferredoxins are useful for in vitro photochemical hydrogen production (Kirk Othmer Chemical Encyclopedia; D. Arnon et al. (1961) Science 134:1425) and for other reactions requiring iron-sulfur proteins as electron carriers, as known to the art. K. Carter et al. (supra) reported numerous differences between the R. japonicum bacteroid ferredoxin and other bacterial ferredoxins. For example, the bacteroid ferredoxin is effective, in the photochemical reduction of acetylene but ineffective as a cofactor in the clostridial phosphoroclastic reaction. Both ferredoxins from A. vinelandii are effective in these reactions. Due to the differences in the redox conditions for the nitrogenase systems of different bacteria, a rhizobial ferredoxin should be used for rhizobial nitrogenase systems, either in vivo or in vitro.
It is therefore desirable to provide a method for the production of rhizobial ferredoxins for use in in vitro hydrogen generation systems and in nitrogenase systems involving rhizobial enzymes. It is also desirable to enhance rhizobial nitrogen fixation by improving electron transport through providing ferredoxin genes to such organisms either to replace defective genes or to add to pre-existing ferredoxin genes. It is also desirable to produce a rhizobial ferredoxin protein as the expression product of a rhizobial ferredoxin gene.